Evaluating intraspecific genetic diversity using environmental DNA and denoising approach: A case study using tank water

Abstract

Recent advances in environmental DNA (eDNA) analysis using high‐throughput sequencing (HTS) provide a noninvasive way to evaluate the intraspecific genetic diversity of aquatic macroorganisms. However, erroneous sequences present in HTS data can result in false positive haplotypes; therefore, reliable strategies are necessary to eliminate such erroneous sequences when evaluating intraspecific genetic diversity using eDNA metabarcoding. In this study, we propose an approach combining denoising using amplicon sequence variant (ASV) method and the removal of haplotypes with low detection rates. A mixture of rearing water of Ayu (Plecoglossus altivelis altivelis) was used as an eDNA sample. In total, nine haplotypes of Ayu mitochondrial D‐loop region were contained in the sample and amplified by two‐step tailed PCR. The 15 PCR replicates indexed with different tags were prepared from the eDNA sample to compare the detection rates between true haplotypes and false positive haplotypes. All PCR replications were sequenced by HTS, and the total number of detected true haplotypes and false positive haplotypes was compared with and without denoising using the two types of ASV methods, Divisive Amplicon Denoising Algorithm 2 (DADA2) and UNOISE3. The use of both ASV methods considerably reduced the number of false positive haplotypes. Moreover, all true haplotypes were detected in all 15 PCR replicates, whereas false positive haplotypes had detection rates varying from 1/15 to 15/15. Thus, by removing haplotypes with lower detection rates than 15/15, the number of false positive haplotypes was further reduced. The approach proposed in this study successfully eliminated most of the false positive haplotypes in the HTS data obtained from eDNA samples, which allowed us to improve the detection accuracy for evaluating intraspecific genetic diversity using eDNA analysis.

Environmental DNA analysis shows high potential as a tool for estimating intraspecific genetic diversity in a wild fish population

“…The tissue DNA was finally eluted in 200 µL of Buffer AE provided in the kit. For Sanger sequencing, almost the entire control region of mitochondrial DNA was amplified using PaaDlp-1_F (5’-GCTCCGGTTGCATATATGGACC-3’) and PaaDlp-1_R (5’-AGGTCCAGTTCAACCTTCAGACA-3’) (Tsuji et al, 2019) by StepOnePlus Real-Time PCR System. The 20-µL reaction mixture contained 900 nM of PaaDlp-1(F/R) in 1 × PCR master mix (TaqMan Gene Expression Master Mix), and 1 µL of tissue-derived DNA (10 ng/µL).…”

“…The field of environmental DNA (eDNA) analysis has rapidly developed over the past decade and has become a useful approach for investigating the species distribution of aquatic macroorganisms (e.g. Ficetola et al, 2008; Jerde et al, 2011; Takahara et al, 2013; Rees et al, 2015; Tsuji et al, 2019). Environmental DNA analysis enables us to detect the presence of species efficiently and non-invasively by analysing eDNA contained in water samples instead of capturing or observing the target species (Rees et al, 2014; Fukumoto et al, 2015; Thomsen et al, 2015; Yamanaka & Minamoto 2016).…”

“…To address the first issue, Tsuji et al (2019) proposed an analytical strategy that combined the denoising of HTS data and subsequent haplotype selection on the basis of detection probability among multiple replications. This study aimed to accurately detect all known haplotypes contained in tank water.…”

“…As a result, regardless of the denoising software package used, the proposed denoising approach successfully eliminated > 99% of false-positive haplotypes derived from PCR and sequencing errors. However, further field studies are still needed to make a robust conclusion on the usefulness of the proposed analytical strategy for evaluating intraspecific genetic diversity because only tank water was used in Tsuji et al (2019).…”

“…Regarding the second issue, to apply eDNA analysis for the evaluation of intraspecific genetic diversity on routine biodiversity surveys, we need to examine how efficiently the eDNA-based methods can evaluate intraspecific genetic diversity in the population at the sampling site. Tsuji et al (2019) reported that we could detect all multiple known haplotypes derived from eDNA of tank water; however, field water usually contains more varying haplotypes with low concentrations, PCR inhibitors, and foreign substances. Thus, there is still uncertainty about the usefulness of the eDNA-based method for evaluating intraspecific genetic diversity from field water.…”